Project 1: Anoctamin8 Tethers the Endoplasmic Reticulum and Plasma Membranes to Assemble Ca2+ Signaling Complexes at ER/PM Junctions. Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junctions, are the sites where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tethers in receptor-evoked Ca2+ signaling is not well understood. Here, we identified Anoctamin 8 (ANO8) as a key tether in the formation of the ER/PM junctions that is essential for STIM1-STIM1 interaction and STIM1-Orai1 interaction and channel activation at a ER/PM PI(4,5)P2-rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2-mediated Ca2+ influx into the ER. This controls the efficiency of receptor-stimulated Ca2+ signaling, Ca2+ oscillations, and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling. These studies have been published in EMBO Journal (2019)38:e101452. Future plans for STIM1, Orai1 and TRPC channels: a) We started examining regulation of STIM1-Orai1 and the ER/PM junctions by lipids. b) We continue to examine how STIM1 gates the TRPC and Orai1 channels. Currently, we are studying the role of the ER/PM junctions in regulation of TRPC channels. c) We are analyzing mice with targeted deletion and overexpression of the Orai1 channel native protein inhibitor SARAF in diseases of secretory glands. d) we are using salivary glands with targeted deletion of AQP5 and NKCC1 in acinar cells and NOD mice with aberrant expression of CFTR in the ducts to determine the role of acinar cell secretion of antimicrobials in the control of the gut and oral cavity microbiomes. Topic B) Intracellular Ca2+ channels (15% effort) Project 2: Lysosome signaling controls the migration of dendritic cells. In collaboration with Hyun-Jin Kim (former postdoc, now Tenured Associate professor at Sungkyunkwan University Medical School, Suwon, Korea) we have reported that the endosomal Ca2+ release channel TRPML3 has a key role in controlling autophagy. We showed that TRPML3 undergoes palmitoylation at its C-terminal region, which is required for dynamic trafficking and cellular function of TRPML3 in autophagy. Palmitoylation regulated TRPML3 surface expression and trafficking, but not channel properties or localization and function of intracellular TRPML3. Activation of intracellular TRPML3 induced robust Ca2+ release, which solely increased autophagy in Ca2+- and palmitoylation-dependent manners. Palmitoylation regulated not only intracellular TRPML3 trafficking to autophagic structures but also autophagic flux in induced autophagy. Importantly, nutrient starvation activated TRPML3 to release Ca2+ and increased the level of TRPML3 palmitoylation. Disruption of TRPML3 palmitoylation, however, abolished the starvation-induced TRPML3 activation without affecting channel activity. These results suggest that trafficking and channel function of TRPML3 are regulated in the context of autophagy, and palmitoylation is a prerequisite for the function of TRPML3 as a Ca2+ channel in autophagosome formation. These studies have been published in Autophagy, 2019;15:327-340. In collaboration with Sandip Patel (UCL, London, England) we described novel inhibitors of the Two-pore channels (TPCs) and their use to determine TPCs in lysosomal functions and Ebola infection. We reported that identification of a number of FDA-approved drugs as TPC pore blockers. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. A screen of more than 1500 compounds yielded dopamine and estrogen receptors as common hits, which inhibited endogenous and expressed TPC2 channels with high potency. Mechanistically, the drugs reduced channels mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. The results expand TPC pharmacology and support a role for TPCs in Ebola virus entry. These studies were published in Biochim Biophys Acta Mol Cell Res. 2019;1866:1151-1161. Future plans for TRPML and TPC channels: We are collaborating with Dr. Freichel (University of Heidelberg) to study regulation of lysosomal and secretory granules Ca2+ signaling by a newly discovered transmembrane protein TMEM63a through the regulation of the TPC channels. Topic C) fluid and HCO3- secretion (35% effort) Project 3: Regulation of Cl- signaling and ion transport by IRBIT-mediated recruitment of multiple kinases and phosphatases. IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion. These studies were published in Science Signaling, 2018;11(554). pii: eaat5018. doi. In collaboration with Ehud Ohana (former postdoctoral fellow, now tenure trac Assistant Professor, Ben Gurion University, Beer Sheva, Israel) we discovered a role for succinate signaling in stone formation (kidney and salivary glands) and hypertension. Although association between stone formation and hypertension is well established, the molecular mechanism linking these pathophysiologies has been elusive. To address this issue, we assessed blood and urine levels of metabolites, renal protein expression, and BP in mice lacking the slc26a6 transporter that inhibits the succinate transporter NaDC-1 to control citrate absorption. Compared with control mice, slc26a6-/- mice had a 40% decrease in urinary excretion of succinate, a 35% increase in serum succinate, and elevated plasma renin. slc26a6-/- mice also showed activity-dependent hypertension that was unaffected by dietary salt intake. Structural modeling, confirmed by